Interactions between microbes and their hosts, including man, depend on specific communication systems. Thus bacteria, including pathogens, perceive their hosts by sensing chemical signals and respond appropriately. Understanding these signaling pathways, and how the microbe and its host respond, could lead to strategies for preventing pathogenesis or fostering beneficial relationships. Agrobacterium tumefaciens, which interacts with plants, provides an excellent model for such studies. As part of this interaction, the bacterium responds to a plant signal by eliciting a second signal that is then perceived by the entire bacterial population. This second quorum-sensing signal controls transfer of the Ti plasmid, a virulence element, to other bacteria. The long term goal is to understand this hierarchical signaling process including how the quorum-sensing signal, called AAI triggers activation of the positive transcription factor, TraR, how TraR retains its activity, and how a specific antiactivator, TraM, interferes with TraR activity. There are three goals for the project period. First we will examine the effect of signal loss on the structure of TraR using genetic screens and biochemical and spectral technologies. We will probe the structure of monomer TraR using a mutant that can bind signal but cannot form dimers. In the second goal we will examine the interaction between TraR and components of RNA polymerase including RpoA and RpoD. Our goal is to identify the amino acids of TraR, of RpoA, and of RpoD that contribute to stable complexes. We will establish conditions for isolating stable complexes of TraR and a C-terminal fragment of RpoD preparatory to efforts to determine the crystal structure of the complexes. In the second goal, we will assess the nature of the interaction between TraR and the antiactivator TraM. We have established a collaboration to determine the crystal structure of TraM and also the crystal structure of the complex formed by TraM and TraR. We also will probe the role of TraM and Lon protease in determining the stability of TraR using physiological tests to measure rates of TraR turnover. We also will establish an in vitro system using purified proteins to characterize the Lon-mediated degradation of TraM. In the third goal we propose to explore the evolutionary diversity of the Ti plasmid quorum-sensing systems by isolating and characterizing plasmids that induce transfer genes in response to novel plant signals.